This invention relates generally to antimicrobial peptides and specifically to a new class of antimicrobial peptides referred to as polyphemusin-like peptides.
In 1981, the self-promoted uptake hypothesis was first proposed to explain the mechanism of action of polycationic antibiotics in Pseudomonas aeruginosa. According to this hypothesis, polycations interact with sites on the outer membranes of Gram-negative bacteria at which divalent cations cross-bridge adjacent lipopolysaccharide molecules. Due to their higher affinity for these sites, polycations displace the divalent cations and, since the polycations are bulkier than the divalent cations, cause structural perturbations in the outer membrane. These perturbations result in increased outer membrane permeability to compounds such as the xcex2-lactam antibiotic nitrocefin, the eukaryotic non-specific defense protein lysozyme and to hydrophobic substances. By analogy, molecules accessing this pathway are proposed to promote their own uptake.
It has been clearly demonstrated that the outer membranes of Gram-negative bacteria are semipermeable molecular xe2x80x9csievesxe2x80x9d which restrict access of antibiotics and host defense molecules to their targets within the bacterial cell. Thus, cations and polycations which access the self-promoted uptake system are, by virtue of their ability to interact with and break down the outer membrane permeability barrier, capable of increasing the susceptibility of Gram-negative pathogenic bacteria to antibiotics and host defense molecules. Hancock and Wong demonstrated that a broad range of such compounds could overcome the permeability barrier and coined the name xe2x80x9cpermeabilizersxe2x80x9d to describe them (Hancock and Wong, Antimicrob. Agents Chemother., 26:48, 1984). While self-promoted uptake and permeabilizers were first described for P. aeruginosa, they have now been described for a variety of Gram-negative bacteria.
Over the past decade, non-specific defense molecules have been described in many animals, including insects and humans. One subset of these molecules have in common the following features: (a) they are small peptides, usually 15-35 amino acid residues in length, (b) they contain four or more positively charged amino acid residues, either lysines or arginines, and (c) they are found in high abundance in the organisms from which they derive. Several of these molecules have been isolated, amino acid sequenced and described in the patent literature (e.g., cecropins: W08900199, WO 8805826, WO8604356, WO 8805826; defensins: EP 193351, EP 85250, EP 162161, U.S. Pat. No. 4,659,692, WO 8911291). However, only limited amounts of these peptides can be isolated from the host species. For example, Sawyer et al. (Infect. Immun. 56:693, 1988) isolated 100-200 mg of rabbit neutrophil defensins 1 and 2 from 109 primed peritoneal neutrophils or lipopolysaccharide-elicited alveolar macrophages (i.e., the numbers present in a whole animal).
Production of these peptides using peptide synthesis technology produces peptides in limited amounts and is expensive when scaled up or when many variant peptides must be produced. Also, structural analysis is difficult without specific incorporation of 15N- and 13C-tagged amino acids which is prohibitively expensive using amino acid synthesis technology.
The hemocytes of the horseshoe crab contain a unique family of xcex2-sheet peptide antibiotics, including polyphemusins I and II and tachyplesins I to III (Nakamura et al. (1988) J. Biol. Chem. 263:16709-16713; and Miyata et al. (1989) J. Biochem. 106:663-668). These peptides are structurally closely-related and are highly abundant in the hemocyte debris. Polyphemusins, isolated from Limulus polyphemus, and tachyplesins, isolated from Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpius rotundicauda, are 18 and 17 amino acid residues in length, respectively. These peptides exhibit a variety of biological activities such as inhibition of the growth of bacteria and fungi and inhibition of the replication of enveloped viruses including vesicular stomatitis virus, influenza A virus and human immunodeficiency virus (HIV)-1 (Miyata et al. (1989) J. Biochem. 106:663-668; Masuda et al. (1992) Biochem. Biophys. Res. Commun. 189:845-850; Morimoto et al. (1991) Chemotherapy 37:206-211; and Murakami et al. (1991) Chemotherapy 37:327-334), herpes virus, hepatitis B and C viruses, and the like. Other studies indicated that tachyplesin I binds to anionic molecules such as DNA and lipopolysaccharides (LPS), and inhibits the LPS-mediated activation of factor I, which is an initiation factor in the Limulus clotting cascade (Nakamura et al., supra; Miyata et al., supra; Yonezawa et al, (1992) Biochemistry 31:2998-3004). Therefore, these arthropod peptides are of special pharmaceutical interest as potential therapeutic agents for anti-endotoxin therapy.
Among the five arthropod peptides, only the secondary structure of tachyplesin I has been determined by nuclear magnetic resonance spectroscopy (Kawano et aL (1990) J. Biol. Chem. 265:15365-15367). It was found to have a fairly rigid planar conformation consisting of an anti-parallel xcex2-sheet structure, constrained by two disulphide bridges and connected by a type II xcex2-turn. In this planar confirmation, five bulky hydrophobic side groups are located on one side of the plane and six cationic side groups are distributed at the xe2x80x9ctailxe2x80x9d of the molecule. Like many naturally occurring antimicrobial peptides, polyphemusins and tachyplesins are polycationic and amphipathic, and the C-terminus is amidated. These properties have been implicated in the mode of action and toxicity of tachyplesin I (Park et al. (1992) Biochemistry 31:12241-12247). Numerous studies of the anti-viral action of this group of peptides against HIV-1 have been carried out (Tamamura et al. (1993) Biochim. Biophys. Acta 1163:209-216, Tamamura et aL (1998) Bioorg. Med. Chem. 6:1033-1041; Arakaki et al. (1999) J. Virol. 73:1719-1723). However few studies have focused on the antimicrobial mechanism and anti-endotoxin activity. Limited data has indicated that, at high concentrations ( greater than 100 fold the inhibitory concentration), tachyplesin I causes morphological and permeability changes of bacterial cells and human erythrocytes, and increases the K+ permeability of S. aureus and E. coli cells, concomitantly reducing cell viability (Katsu et al. (1993) Bio. Pharm. Bull. 16:178-181).
Gram-negative bacteria have two cell envelope membranes. The outer membrane is an asymmetric membrane with the bulky glycolipid lipopolysaccharide (LPS) covering more than 90% of the cell surface in its outer leaflet, and phospholipids with a composition similar to that of the cytoplasmic membrane in its inner leaflet. Many antimicrobial cationic peptides have been shown to interact with the LPS of the Gram-negative bacterial outer membrane and pass across this membrane by self-promoted uptake, followed by interaction with and insertion into the negatively charged cytoplasmic membrane (Hancock (1997) Lancet 349:418-422). However, the target of these cationic peptides is not well understood. Although for many peptides the formation of lesions has been observed in model membranes, there has been little convincing evidence to link such interactions to the event(s) causing bacterial cell death, and it has been proposed that at least some peptides cross the cytoplasmic membrane to access cytoplasmic targets like polyanionic nucleic acids (Kagan et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:210-214; Ludtke et al. (1996) Biochemistry 35:13723-13728).
There is thus a need to develop polypeptides having a broad range of potent antimicrobial activity against a plurality of microorganisms, including gram negative bacteria, gram positive bacteria, fungi, protozoa, viruses and the like.
The present invention provides cationic peptides, referred to as polyphemusin-like peptides, which have antimicrobial activity. Also included are analogs, derivatives and conservative variations thereof.
In a first embodiment, the invention provides an isolated peptide having an amino acid sequence selected from the group consisting of: WCFZ5VCZ2RGZ3CRZ2KCRR, Z2RWCFRVCYZ3GZ2CZ3Z5Z2CR, RRWCFZ5VCZ3RGZ4CYZ4Z4CRZ1, RZ5WCZ3Z2Z3CYRGFCZ3Z2Z5CR, RRWCZ3RVCYZ5GFCYRKCR, and RRWCFRVCYRGZ3FCYRKCR; wherein Z1 is a basic amino acid residue or no amino acid residue; Z2 is a basic or aromatic residue, Z3 is a basic amino acid residue, Z4 is arginine, valine or alanine, and Z5 is an aromatic or aliphatic amino acid residue. Exemplary peptides of these general formulae include SEQ ID NO:1 to SEQ ID NO:11.
The invention also provides a method of inhibiting the growth of microbes such as bacteria and yeast, comprising contacting the bacteria or yeast with an inhibiting effective amount of a peptide having an amino acid sequence selected from the group consisting of WCFZ5VCZ2RGZ3CRZ2KCRR, Z2RWCFRVCYZ3GZ2CZ3Z5Z2CR, RRWCFZ5VCZ3RGZ4CYZ4Z4CRZ1, RZ5WCZ3Z2Z3CYRGFCZ3Z2Z5CR, RRWCZ3RVCYZ5GFCYRKCR, and RRWCFRVCYRGZ3FCYRKCR; wherein Z1 is a basic amino acid residue or no amino acid residue; Z2 is a basic or aromatic residue, Z3 is a basic amino acid residue, Z4 is arginine, valine or alanine, and Z5 is an aromatic or aliphatic amino acid residue, alone, or in combination with an antibiotic. Exemplary peptides used in the practice of invention method are peptides having the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:11. Peptides of the invention can be administered in combination with antibiotics, lysosymes, anti-TNF (tumor necrosis factor) antibodies and TNF antagonists. Classes of antibiotics which can be used for synergistic therapy with the peptides of the invention include aminoglycoside, penicillin, cephalosporine, fluoroquinolone, carbepenem, tetracycline and macrolide.
In another embodiment, the invention provides a method of inhibiting an endotoxemia or sepsis associated disorder in a subject having or at risk of having such a disorder, comprising administering to the subject a therapeutically effective amount of a peptide of the invention.